7. PROJECT SUMMARY The long-term goal of the proposed research is gain fundamental insights into the process of tooth replacement. We hope to learn how the dentition is patterned across the jaw and to identify some of the key molecules and cells required to kick-start tooth replacement in adult tissues. In the future, our work will form the basis for bioengineering of tooth replacements. The project is specifically aimed at studying tooth replacement in a non-traditional animal model, the leopard gecko because this animal, unlike mouse, can replace its teeth throughout life. The rationale for choosing this particular lizard is as follows: 1) reptiles and mammals are amniotes and thus share a common ancestor. Therefore discoveries are more likely to be applicable to human than those on fish or the derived mouse dentition, 2) adult geckos have large numbers of teeth that are replaced throughout life, 3) gecko teeth are only attached to bone on the labial side, leaving the lingual successional lamina accessible to experimental manipulation, 4) the tooth replacement cycle is much shorter than other reptiles such as alligators and iguanas so phenotypes will be visible sooner, 5) leopard geckos are readily available from pet suppliers and animals are easy to handle. 6) we have already studied this animal extensively and have characterized the shedding and eruption of teeth over time. The gecko offers unique access to the developing adult dentition compared to mouse, fish and shark models. Specifically, we have developed ways to serially record the whole dentition while the animals are still alive including taking miniature wax bites and photographing fluorescent teeth that were labelled with a special compound taken up by mineralizing tissues. The ability to monitor the effects of dental surgery to the dental lamina in living animals is not possible in other models. We will use this grant to optimize surgery and drug delivery systems to study the adult gecko dentition in 4D. The aims are: 1) to use surgery to disrupt the dental lamina and partially formed dentition in order to isolate the key cells required for tooth initiation. 2) To manipulate the WNT signaling pathway during in vivo tooth replacement and then to study the effect on patterning of the dentition, proliferation and downstream gene expression changes. Innovative methods of animal anesthesia will permit precision surgical procedures to be carried out such as bead implants. The synergistic research environment consists of the PI who is a pediatric dentist and two post-doctoral fellows with backgrounds in dental development and evolution.